A major challenge for all cell therapies is the need to develop cost-effective and efficient manufacturing and delivery capabilities, such as creating a scalable, automated closed system to isolate a subset of T cells. For example, studies in mice and humans show that the infusion of less differentiated T cells that are genetically engineered with chimeric antigen receptor (CAR) or T cell receptor (TCR) leads to superior cell expansion, persistence, and cancer elimination. Sorting and isolating a subset of T cells (e.g. naïve CD8+ cells) requires multiple markers (e.g. CD8, CD62L & CD45RA), which is challenging to scale up and automate.
FACS (fluorescence activated cell sorting) is the product most used for the detailed analysis of individual cells. Using FACS, it is difficult to scale up for bulk isolation of cells. Magnetic cell sorting (MACS) techniques, particularly the Miltenyi system, are used for targeted cell isolation in bulk. Cells bound to microbeads (e.g. Dynabeads) can be pulled out by an external magnet outside the mixing tube. However, binding antibody-conjugated microbeads to cells, rather than antibodies alone or antibody-conjugated nanobeads, slows down the process. Therefore, most magnetic cell sorting systems on the market use nanobead over microbead-conjugated antibodies. One of the biggest problems with MACS is that isolated cells coupled to magnetic microparticles (>1 μm) are often destroyed.
Existing MACS technology applies high-gradient magnetic cell separation columns to magnetize nanobead-labeled cells in a magnetic field, generated by a strong external magnet. MACS uses ferromagnetic steel-wool filled columns to strengthen the long-distance interaction between the low magnetic nanobeads and the external magnet. However, the use of the steel-wool packed column in this system creates other technical problems like column clogging and increased manufacturing costs. Moreover, the MACS nanobeads stick to the targeted cells, either on the cell surface or inside by endocytosis. This limits the application of MACS primarily to cell enrichment based on a single surface marker.
As described in the International Patent Application published under the publication number WO 2015/175344 A1, a BUBLES (buoyancy enabled separation) or buoyancy activated cell sorting (BAGS) technology by applying targeted microbubbles for cell isolation has been developed for cell isolation.
For multi-parameter MACS, the prior art contains a few methods enabling the reversible binding of cells and affinity ligand-conjugated magnetic beads. These were developed by sequentially targeting multiple cell-surface markers. For example, the Streptamer technology enables the reversible target cell binding of magnetic microparticles (˜1 μm) by conjugating to Strep-Tactin multimerized low-affinity single-chain antibodies that have been engineered to fuse with a Strep-tag. Other technologies in the prior art use modified antibodies that can dissociate from cells or magnetic beads. These methods usually require extensive modifications to the antibodies of interest and can be applied to other platforms beyond MACS, such as FACS, BAGS and other forms of affinity purification.
WO 2015/175344 A1 teaches a BACS method for clinical applications, including the isolation of circulating tumor cells and cord blood stem cells. This technology has several advantages over other prior art in performing bulk cell isolation. First, its lipid shell microbubble is self-molding to external forces (e.g. ultrasound and bound cells) because it is the most compressible and flexible shell-class microbubbles (others including albumin, polymer, and glass). In conjunction with a gas core, it is a very gentle material for cell isolation. Second, the microbubble it uses is a “self-driving vehicle”, as microbubble-bound cells automatically float to the top surface of a liquid. Third, the lipid-shelled microbubbles it uses self-separate from water-based solutions and self-concentrate/aggregate to other microbubbles. Fourth, users of this technology working with cell-bound microbubbles can disrupt their internal bonds, without causing cell damage, by increasing ambient air pressure.
Emulsification is used to prepare perflorohexane gas microbubbles (MBs) within phospholipid shells. DSPE-PEG 3400-maleimide is used in the microbubble membrane to conjugate antibodies to the microbubbles. It uses Fc fragment-specific IgG carrying 1-2 thiol groups per antibody to conjugate antibodies onto maleimide-activated MBs via Michael addition. The technology uses targeting antibodies, such as anti-EpCAM, the most widely accepted marker for isolating circulating tumor cells of epithelial origin for coupling. Most the MBs in this process have a diameter of 3-10 μm. Each MB has, on average, 367,000 anti-Fc IgG molecules.
BAGS (or BUBLES) is an innovative cell isolation/sorting platform that can be used alone or in combination with the other two major platforms in the prior art (FACS and MACS) for challenging tasks. In its preferred embodiment, the present disclosure uses BACS for the bulk isolation, multiple-marker sorting, and manipulation of human cells in a single container device. In other embodiments, the present disclosure uses BACS in conjunction with FACS or MACS for this isolation, sorting, and manipulation process.
Cell based therapy is a rapidly developing medicine of intense research and great potential to patient benefits. The majority of cell therapies in clinical trials today are hematopoietic or mesenchymal stem cells and immune T cells for genetic diseases and cancers, these targeted cells have common and well known characteristics of being very rare subsets and the need of multiple cell surface markers for specific isolation from the heterogeneous cell mixtures in blood or human tissues, and of being very difficult to regulate cell differentiation progress or maintain cell sternness during isolation, expansion and in vitro manipulation procedures. This invention is intended to overcome a number of these obstacles.